Regeneration of sinus node〜 reconstitution of biological pacemaker using bio-nanotechnololgy

我们使用一种新技术构建了心脏起搏器薄片，该技术带有细胞排列的模式。制备了天然杀伤细胞-1抗体标记的磁性纳米颗粒(直径~10 nm)，用于标记大鼠心脏特殊传导系统。从表达GFP的大鼠胚胎(14.5d.p.c.)中分离出心肌细胞(CM)。与AM共同孵育，分散在多电极培养皿中。在培养皿下放置一个小磁铁以排列标记的CM(MAG组，n=5)。对照组(CONT组，n=5)。3天后，细胞外电位恢复正常。记录以检查起搏和传导特性。将MAG组培养的CM移植到培养的新生大鼠CM上。3天后，供体(胚胎)和受体(新生)CM均被负载Rhod2，并用高速CCD摄像机记录[Ca~(2+)]i瞬变以评估供体和受体CM之间的电相互作用。在MAO中，AM标记的CM聚集在磁铁上，而在CONT中，AM标记的CM随机分布在未标记的CM中。细胞外电位记录显示，来自AM标记的CM聚集的规则自发兴奋在周围未标记的CM上有规律地传播。相比之下，CONT表现出相当大的好奇心，起搏部位与包括螺旋型折返在内的不规则传播有关。此外，MAG组的自发搏动频率明显快于CONT组(16±3vs11±2次/分，p&lt；0.05，n=5)。加入异丙肾上腺素(1μM)可增加MAG和CONT的自发搏动频率。[Ca^2+]；在MAG中，移植的胚胎CM和受体新生儿CM的瞬变具有良好的同步性。利用磁性纳米颗粒构建细胞排列模式可能是一种新的技术，可以设计出组织良好的心瓣膜，以创建可靠的生物起搏器。

We constructed cardiac pacemaker sheets using a novel technique with patterning of cell alignment. Natural killer cell-1 antibody-conjugated magnetic nanoparticles (AM) which contains magnetite nanoparticles(diameter〜10 nm), was prepared to label rat cardiac specialized conduction system. Cardiomyocytes(CM) isolated from the GFP expressing rat embryo(14.5d.p.c.) were incubated with AM and dispersed on a culture dish with 64 multielectrodes. A small magnet was placed under the dish to arrange labeled CM (MAG group, n=5). CM cultured without magnet were referred as control(CONT group, n=5). After 3 days, extracellular potentials were. recorded to examine pacing and conduction properties. Cultured CM of MAG group were transplanted on a sheet of cultured CM from neonatal rats. Three days later, both donor(embryo)and recipient(neonate)CM were loaded with Rhod2 and [Ca2^+]i transients were recorded using a high-speed CCD camera to estimate electrical interaction between donor and recipient CM. In MAO the AM-labeled CM were aggregated over a magnet, whereas in CONT, the AM-labeled CM were randomly distributed among the unlabeled CM. Extracellular potentials recording revealed that regular spontaneous excitation originating from the aggregation of AM-labeled CM propagated regularly over surrounding unlabeled CM. CONT, in contrast, showed considerable wondering of pacemaker sites in association with irregular propagations including spiral-type reentry. In addition, spontaneous beating rates in MAG were significantly faster than CONT(16±3 vs 11±2 bpm, p<0.05, n=5). The addition of isoproterenol(1μM) increases the spontaneous beating rate in both MAG and CONT. [Ca^2+] ; transients of transplanted embryonic CM and recipient neonatal CM were well synchronized in MAG The patterning of cell arrangements using magnetic nanoparticles could be a novel technique to engineer well-organized cardiac sheets for creation of dependable biological pacemaker.